Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how he reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events -- which are not understood mechanistically -- will elucidate interactions and rearrangements among core components and may serve as a paradigm for other rearrangements and multiple recognition events that occur elsewhere in the spliceosome. This proposal focuses first on the first ATP-dependent step of spliceosome assembly - the stable binding of U2 snRNP around the branch region, and investigates the action of an ATPase, Prp5. As the first ATP-dependent event, this step provides a uniquely simplified system, for studying the action of a spliceosomal ATPase. Further experiments will focus on the mechanism and the consequences of a recently identified bridging interaction between U1 and U2 snRNPs that is mediated by Prp5. Finally, using a new genetic screen, we are investigating interactions between the branch site (and the 5' and 3' splices sites) and components of the spliceosome critical for the second catalytic reaction, interactions between the identified components and the RNA substrate, and interactions of the identified components with other constituents of the spliceosome -- with a particular bent as to mechanism by which these components interact to help juxtapose the reactants for the second chemical step.